Arabidopsis Protein Phosphatase 2C ABI1 Interacts with ... - Cell Press

Molecular Plant 7, 960–976, June 2014

RESEARCH ARTICLE

Arabidopsis Protein Phosphatase 2C ABI1 Interacts with Type I ACC Synthases and Is Involved in the Regulation of Ozone-Induced Ethylene Biosynthesis Agnieszka Ludwikówa,1, Agata Cieślab, Anna Kasprowicz-Maluśkic, Filip Mitułaa, Małgorzata Tajdela, Łukasz Gałgańskia, Piotr A. Ziółkowskia, Piotr Kubiakd, Arleta Małeckae, Aneta Piechalake, Marta Szabata, Alicja Górskaa, Maciej Dąbrowskia, Izabela Ibragimowa, and Jan Sadowskia,b a Department of Biotechnology, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznań, Umultowska 89, 61–614 Poznań, Poland b Institute of Plant Genetics Polish Academy of Science, Strzeszyńska 34, 60–479 Poznan, Poland c Department of Molecular and Cellular Biology, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznań, Umultowska 89, 61–614 Poznań, Poland d Department of Biotechnology and Food Microbiology, University of Life Sciences, Wojska Polskiego 48, 60–627 Poznań, Poland e Department of Biochemistry, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznań, Umultowska 89, 61–614 Poznań, Poland

ABSTRACT Ethylene plays a crucial role in various biological processes and therefore its biosynthesis is strictly regulated by multiple mechanisms. Posttranslational regulation, which is pivotal in controlling ethylene biosynthesis, impacts 1-aminocyclopropane 1-carboxylate synthase (ACS) protein stability via the complex interplay of specific factors. Here, we show that the Arabidopsis thaliana protein phosphatase type 2C, ABI1, a negative regulator of abscisic acid signaling, is involved in the regulation of ethylene biosynthesis under oxidative stress conditions. We found that ABI1 interacts with ACS6 and dephosphorylates its C-terminal fragment, a target of the stress-responsive mitogen-activated protein kinase, MPK6. In addition, ABI1 controls MPK6 activity directly and by this means also affects the ACS6 phosphorylation level. Consistently with this, ozone-induced ethylene production was significantly higher in an ABI1 knockout strain (abi1td) than in wild-type plants. Importantly, an increase in stress-induced ethylene production in the abi1td mutant was compensated by a higher ascorbate redox state and elevated antioxidant activities. Overall, the results of this study provide evidence that ABI1 restricts ethylene synthesis by affecting the activity of ACS6. The ABI1 contribution to stress phenotype underpins its role in the interplay between the abscisic acid (ABA) and ethylene signaling pathways. Key words: hormonal regulation; protein phosphorylation/dephosphorylation; signal transduction; Arabidopsis. Ludwików A., et al. (2014). Arabidopsis protein phosphatase 2C ABI1 interacts with type I ACC synthases and is involved in the regulation of ozone-induced ethylene biosynthesis. Mol. Plant. 7, 960–976.

Introduction Ethylene is an essential signaling molecule involved in plant growth, development, and stress responses (Wang et al., 2002; Stepanova and Alonso, 2009; Tsuchisaka et al., 2009). Under stress conditions, the rate of ethylene production increases rapidly and leads to the activation of cellular responses through ethylene signaling and interactions with other hormone signaling pathways (Overmyer et al., 2000; Wang et al., 2002), the best studied of which is the interplay between ethylene and abscisic acid (ABA) (Ghassemian et al., 2000; Sharp, 2002; Sharp

and LeNoble, 2002; Trivellini et al., 2011). Nevertheless, understanding of ABA and ethylene interaction is still in its infancy.

To whom correspondence should be addressed. E-mail ludwika@ amu.edu.pl, tel. (48) 61 8295964, fax (48) 61 8295949. © The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi:10.1093/mp/ssu025, Advance Access publication 17 March 2014 Received 25 November 2013; accepted 4 March 2014 1

Molecular Plant Ethylene is synthesized in a two-step reaction catalyzed by 1-aminocyclopropane 1-carboxylate synthases (ACSs) and ACC oxidases (Kende, 2001; Wang et al., 2002). ACS (Enzyme Commission (EC) number 4.4.1.14) catalyzes the conversion of S-adenosyl-L-methionine to 1-aminocyclopropane-1-carboxylic acid (ACC). ACC oxidase (EC 1.14.17.4) then catalyzes the conversion of ACC to ethylene with the release of CO2 and cyanide. The formation of ACC is considered to be the rate-limiting step in ethylene biosynthesis (Adams and Yang, 1979; Tsuchisaka et al., 2009). Despite the chemical simplicity of ethylene biosynthesis, the process is strictly regulated at multiple steps. It is accepted that, in addition to transcriptional regulation (Tsuchisaka and Theologis, 2004; Li et al., 2012), posttranslational regulation is crucial for developmental and stress-induced ethylene production (Christians et al., 2009; Han et al., 2010a; Prasad and Stone, 2010; Skottke et al., 2011; Lyzenga et al., 2012). Previous studies provide convincing evidence that the regulation of ethylene synthesis results from stabilization of ACS proteins via the key C-terminal motif (Tatsuki and Mori, 2001; Liu and Zhang, 2004; Joo et al., 2008). Based on the presence or absence of this region, ACS isozymes are divided into three types. Type I ACSs have the longest C-terminal sequences, and these include target motifs for both mitogen-activated protein kinases (MAPKs) and possibly also calcium-dependent protein kinase (CDPK). Type II isozymes contain the putative CDPK phosphorylation sites, whereas type III lack both MAPK and CDPK sites (Yamagami et al., 2003; Sebastià et al., 2004). A significant role for C-terminal extensions in regulating ACS stability has been reported for type I isoforms (Liu and Zhang, 2004; Kamiyoshihara et al., 2010; Choudhury et al., 2012). In Arabidopsis, the phosphorylation of ACS2 and ACS6 by stress-induced MAPKs (MPK3 and MPK6) decreases protein turnover, leading to increased ethylene production (Liu and Zhang, 2004; Joo et al., 2008). Conversely, dephosphorylation immediately targets ACS for degradation via the ubiquitin/26S proteasome pathway (Joo et al., 2008). Furthermore, in a recent study, Arabidopsis RCN1-containing PP2A complexes were found to reduce the accumulation of type I isozymes and to prevent type II turnover (Skottke et al., 2011). Consistently with these findings, the regulation of MPK6 activity is also crucial for the regulation of ethylene production. Schweighofer et al. (2007) demonstrated that negative regulation of MPK6 activity by the protein phosphatase AP2C1 affects wounding-induced ethylene production. In addition to AP2C1, other protein phosphatases have also been implicated in MPK6 regulation (Ulm et al., 2001, 2002; Leung et al., 2006; Brock et al., 2010). One of them is the ABI1 protein phosphatase 2C, a negative regulator of ABA signaling (Gosti et al., 1999; Miyazono et al., 2009), which is known to impose a restriction on MPK6 activity (Leung et al., 2006). However, the biological function of the

The Role of ABI1 in Ethylene Biosynthesis

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ABI1–MPK6 complex and its regulation are still unclear. Other important interacting partners have been also proposed for ABI1 including SnRK2, AtHB6, or SWI3B (Himmelbach et al., 2002; Yoshida et al., 2006; Saez et al., 2008). Previous results showed that, under ozone stress conditions, an ABI1 knockout mutant (abi1td) produced significantly more ACC than wild-type (WT) plants, suggesting that ABI1 plays a specific role in regulating ethylene production during ozone stress (Ludwików et al., 2009). In this study, we demonstrate that ABI1 protein phosphatase, a core component in the ABA signaling pathway, has a significant fine-tuning function in the regulation of ethylene biosynthesis under ozone. We explored the regulatory mechanism by which ABI1 affects the activity of type I ACS isozymes and found that ABI1 directly dephosphorylates ACS6 and negatively regulates MPK6 activity. We also discovered that ABI1 acts to increase the cellular redox state to overcome the detrimental effect of ethylene overproduction. Therefore, we conclude that ABI1 is required to restrict ethylene production under ozone stress conditions.

RESULTS ABI1 Interacts With and Dephosphorylates the C-Terminal Region of ACS6 We have previously shown that full-length ABI1 potentially interacts with full-length ACS2 and ACS6 in yeast (Ludwików et al., 2009). In addition, it has been demonstrated that the stability of type I ACS isozymes is regulated by phosphorylation of the MAPK site (Liu and Zhang, 2004). Therefore, to determine whether ABI1 interacts with ACS at the MAPK site, ACS catalytic domain deletion constructs (∆–ACS2 and ∆–ACS6) were generated and tested for interaction with the ABI1 phosphatase domain construct (∆N– ABI1) in a yeast two-hybrid assay. As shown in Figure 1A, the C-terminal fragment of ACS6 was sufficient for interaction with ABI1, and no interaction between ABI1 and the C-terminal fragment of ACS2 was found. These suggested that ABI1 may be involved in ACS6 dephosphorylation. Interestingly, when we tested for interaction between the homologous phosphatase ABI2 and either ACS2 or ACS6, ABI2 was found to bind to ACS6 only. Bimolecular fluorescence complementation (BiFC) experiments using ABI1 (or ABI2) together with ACS6 also revealed that both ABI1 and ABI2 PP2Cs interact with ACS6 (Figure 2A). No interactions were detected for the ABI1–ACS2 and ABI2– ACS2 pairs. However, treatment with MG132 enabled the ABI1–ACS2 complex to be detected (Figure 2A). No fluorescence was observed in control cells carrying combinations of YFP fusions, empty vector, or the empty vectors alone (Supplemental Figure 1).

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Figure 1 ABI1 Interacts with the C-Terminal Fragment of ACC Synthase 6 in Yeast. Interaction assay of ABI1 and ABI2 PP2Cs with full-length and the C-terminal fragment of ACS proteins. (A) Saturated cultures of the double-transformed yeast cultures were grown on double (DDO—SD medium without leucine and tryptophan), triple (TDO—SD medium without leucine, tryptophan, histidine), and quadruple (QDO—SD medium without leucine, tryptophan, histidine, adenine) selective medium. Representative examples are shown. (B) Schematic structures of ABI1, ABI2, and ACS proteins. The N-terminal deletion of ABI1 (∆N–ABI1) lacks amino acid residues 1–121. ∆–ACS2 and ∆–ACS6 lack the catalytic domain and comprise amino acid residues 464–496 and 471–495, respectively. Deleted protein fragments are indicated by open bars.

To provide further evidence for ABI1–ACS interaction, binding of ABI1 to ACS2 and ACS6 was analyzed using pulldown assays. StrepTag–ABI1 or StrepTag–ABI2 purified from plant extracts were incubated with recombinant GST–ACS2, GST–ACS6, or GST alone. We found that both ABI1 and ABI2 were capable of binding ACS6, but only ABI1, and not ABI2, bound ACS2 (Figure 2B). Finally, to provide evidence that the C-terminal fragment of ACS6 provides a substrate for ABI1, we performed an in vitro dephosphorylation assay. Because ABI1 does not interact with the C-terminal fragment of ACS2 (Figure 1A), only ACS6 was included in this analysis. For the assay, recombinant GST–ACS6, 32P-labeled at the C-terminus by immunoprecipitated MPK6, was used. Following MPK6 inactivation, GST–ACS6 was incubated in the presence of StrepTagged ABI1 from H2O2- and ABAtreated Arabidopsis cells. Clearly, the addition of active ABI1 phosphatase decreased the level of ACS6 phosphorylation (Figure 2C and 2D). Addition of ABA-inhibited ABI1 did not alter the ACS6 phosphorylation level. The phosphatase activity of ABI1 complexes was verified with a non-radioactive phosphatase assay (see Figure 3A). Finally, to further investigate the role of ABI1 in regulation of ACS6, we compared the turnover of ACS6 using a cell-free degradation assay. Recombinant GST–ACS6 was incubated with plant extracts prepared from abi1td or WT protoplasts treated with or without MG132. We observed a slower GST–ACS6 degradation in abi1td extracts compared to WT (Figure 2E). Overall, these results suggest that ABI1 regulates the accumulation of ACS6 protein.

ABI1 Regulates ACS Phosphorylation by Restricting MPK6 Activity In Arabidopsis, ACS2 and ACS6 have been shown to be phosphorylated by MPK6 (Liu and Zhang, 2004). In addition, Leung et al. (2006) demonstrated that ABI1 interacts with MPK6 and inhibits its kinase activity. These results suggest that ABI1 could affect the phosphorylation status of ACS6 by restricting MPK6 activity. To further analyze whether MPK6 deactivation depends on ABI1, we performed an in vivo kinase assay in Arabidopsis protoplasts in which MPK6 activity was monitored using MBP as a substrate. When co-expressed with ABI1, MPK6 was inactivated, but was not affected by ABI2 (Figure 3B and 3D). An in vitro phosphatase assay using active MPK6 complexed with StrepTag–ABI1 and recombinant GST–ABI1 confirmed the in vivo results. As negative controls, GST-tagged protein phosphatase PP2C6, and ABI1 inhibited by ABA were used. The composition of ABI1 complexes used for the repression of MPK6 kinase activity was also analyzed using LC–MS/MS (see Supplemental Data 1–4). Remarkably, reduced kinase activity was observed for MPK6 complexed with either ABI1 from H2O2-treated Arabidopsis cells or recombinant ABI1 (Figure 3C and 3E). Overall, these results support the notion that ABI1 has a specific inhibitory effect on MPK6 activity. Finally, to test whether ABI1 affects ACS phosphorylation via MPK6, we combined ABI1 with active MPK6, and the kinase activity was monitored directly on recombinant

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Figure 2 ABI1 Interacts with ACS6 and Dephosphorylates Its C-Terminal Fragment. (A) Analysis of the interaction between ABI1 and ABI2 with ACS2 and ACS6 by means of a BiFC assay in control (left) and MG132-treated (right) Arabidopsis thaliana protoplasts. Treatment with MG132 for 6 h increases the BiFC signal for the ABI1–ACS2 interaction. The YFP fluorescent signal was located in the cytoplasm. Scale bar: 10 μm. (B) Pull-down assays to verify the interaction of ABI1 with ACS2 or ACS6 (upper panel), interaction of ABI2 with ACS2 or ACS6 (middle panel), and the GST/Strep fusion negative controls (lower panel). Input lines (upper/middle panels, lines 1 and 4; control panel, lines 2 and 3) represent 50% of the ABI1/2. Recombinant GST–ACS2, GST–ACS6 were pre-coupled to glutathione sepharose and incubated with StrepTag–ABI1 and StrepTag–ABI2, respectively. Pulled-down StrepTagged ABI1 or ABI2 proteins were detected (IB) with the epitope tag antibody. The presence of recombinant protein was confirmed using specific anti-ACS2 or anti-ACS6 antibodies. Note, for StrepTag–ABI1 isolated from T87 cell-suspension cultures, two sharp protein bands were observed that may arise from unknown protein modification. (C) 32P-labeled GST–ACS6 was mixed with 3 μg of StrepTag–ABI1 purified from Arabidopsis T87 cell cultures subjected to various treatments. ACS phosphorylation level was visualized by autoradiography. ABA-inhibited StrepTag–ABI1 was used as a negative control. Western blot with anti-GST antibodies confirms the presence of the GST–ACS6 protein. The experiment was performed twice, with consistent results.

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GST–ACS2 or GST–ACS6 as a substrate. As controls, we used the protein complex assembled in the presence of StrepTagged ABI2 and ABA-inactivated ABI1. Figure 3F shows that MPK6 inactivation by ABI1 resulted in reduced ACS2 and ACS6 phosphorylation. As expected, MPK6induced ACS2 phosphorylation was not affected by ABI2 and ABA-inactivated ABI1. Overall, these results demonstrate that ABI1 influences ACS phosphorylation by regulating MPK6 activity.

Molecular Plant MG132-treated abi1td mutant (Figure 4D). Moreover, in vivo ACC oxidase activity was not affected in the abi1td mutant (Figure 4E), consistently with the increase in ethylene biosynthesis in abi1td depending on the ACS6 isozyme. Overall, these observations corroborate the role of ABI1 in the regulation of ethylene biosynthesis.

Increased Ethylene Production in the abi1td Mutant in Response to Ozone Treatment

An ABI1 Knockout Strain Shows Elevated Ethylene Levels and ACS Activity in Response to Chemical Treatment Our results suggest that ABI1 regulates ACS6 by direct dephosphorylation and by inhibition of MPK6 activity. Therefore, we expected that an increase in ACS6 activity causes ethylene overproduction in an ABI1 loss-of-function mutant. To verify this, WT Col-0 and abi1td seedlings were treated with CuSO4 and the proteasome inhibitor, MG132. In this study, the abi1td mutant used (Ludwików et al., 2009) corresponds to the abi1-3 allele isolated by Saez et al. (2006). Copper ions are known to induce ethylene production via an increase in ACC synthase activity and by upregulation of ACS genes (Arteca and Arteca, 1999; Jonak et al., 2004). MG132 is commonly used to block the proteolytic activity of the 26S proteasome complex and has been shown to induce apoptotic cell death through formation of reactive oxygen species (Han et al., 2010b). As expected, the abi1td mutant and WT Col-0 showed comparable (not statistically different) ethylene levels under normal conditions (Figure 4A and Supplemental Figure 2). However, when treated with CuSO4, we observed an upsurge in ethylene production in the abi1td mutant (p